There has been rapid growth in recent years in apparatus and methodology for biochemical enterprise, particularly for cleavage, separation and analysis of macromolecules such as DNA, including human DNA. There are a number of uses, such as determining the presence of genetically induced conditions such as Sickle-Cell Anemia, Huntington's Chorea, and others. Fluorescent gel scanning has also become important as an identification tool. Genetic code from a human hair or a flake of skin can be matched to a single human being, supplementing older procedures, such as fingerprint analysis in criminal law.
Macromolecules are typically separated and identified by electrophoresis. In electrophoretic separation electrically charged macromolecules of different size and structure move at different velocities in a specific medium under the influence of an electrical field. Typical mediums and geometries in use include agarose slabs, polyacrylamide gel films, matrix-filled columns, and micro columns (capillary tubings) either with or without a gel matrix.
The use of micro-column, (capillary) electrophoresis for macromolecule separation and analysis is a relatively more recent development than slab and film techniques. A principle advantage of micro-column techniques is their suitability for analysis of very small sample volumes, e.g. microliter or sub-microliter amounts of sample. Being able to analyze such small amount of sample has become very important because, oftentimes, the available volume of sample for analysis is extremely small. For example, in forensic and criminal investigations, the only material that may be available for analysis may comprise a fingernail scraping or a stain on an article of clothing.
Another advantage of microcolumn techniques is that the relatively smaller and simpler apparatus lends itself to techniques of automation more readily than do slab and film systems. For example, a detector to monitor bands passing a point in a micro-column is a relatively simple device compared to a scanning detector for monitoring a plurality of channels in an agarose slab. The column itself is small and easier to manipulate. The reservoirs for storing buffers and other solutions are smaller as well and more easily managed. There are many other examples of the advantages of micro-column electrophoresis.
With all of the advantages and advances in electrophoresis by the use of micro-columns, there remain problems that have not been solved before the present invention. Many of these are simply variations of difficulties encountered in the effort to automate all types of electrophoresis apparatus. For example, considering only capillary electrophoresis, not all starting samples respond in the same way to the same set of electrophoretic circumstances. By circumstances is meant the value set of all of the variables that effect electrophoresis of sample material.
The variable set includes the length and bore of the capillary, the separation matrix in the column, the electrical potential maintained across the ends of the column, the types of buffers used, the geometry of the physical apparatus, the arrangement and techniques for sample injection, ability to control temperature and to dissipate heat generated in the electrophoresis operations, arrangements for fraction collection and more.
Variable sets for specific samples and purposes have been developed empirically over time and tested. New circumstances for new sample compositions continue to be developed.
Some circumstances are relatively easy to manage. For example, the electrical potential across the ends of a column. Others are quite difficult. The only way to change the capillary bore, for example, is to change the capillary, and to do so is a relatively clumsy operation in many existing systems.
What is clearly needed is an apparatus wherein the circumstances difficult to change are incorporated into a module that can be placed in a serving apparatus to provide the circumstances more generic and relatively easier to manage. In this invention, the module would provide circumstances specific to a pretested specification, for separations of a certain type and class of sample. The module, which is called an application-specific cassette by the inventors, includes a support block, or framework, designed to interface physically to the serving apparatus; a capillary of the correct length, bore, material, and wall thickness, and incorporating a particular gel or liquid matrix; buffers solutions at the ends of the capillary; and one or more reservoirs for liquids needed in the specific procedure. In a specific and preferred embodiment of the present invention, the application-specific cassette is disposable. When the buffers are depleted, the matrix ages and deteriorates, or any other change renders the cassette unusable, the cassette is simply removed, and a new one substituted to the serving apparatus.
The serving apparatus includes a robotic system with a liquid-handling apparatus having a probe translatable in three dimensions, and an electrode in contact with fluid in the liquid handling apparatus.